Vascular smooth muscle ROCK1 contributes to hypoxia-induced pulmonary hypertension development in mice.

Rho kinase (ROCK) is implicated in the development of pulmonary arterial hypertension (PAH) in which abnormal pulmonary vascular smooth muscle (VSM) contractility and remodeling lead to right heart failure. Pharmacologic ROCK inhibitors block experimental pulmonary hypertension (PH) development in rodents but can have off-target effects and do not distinguish between the two ROCK forms, ROCK1 and ROCK2, encoded by separate genes. An earlier study using gene knock out (KO) in mice indicated that VSM ROCK2 is required for experimental PH development, but the role of ROCK1 is not well understood. Here we investigated the in vivo role of ROCK1 in PH development by generating a VSM-targeted homozygous ROCK1 gene KO mouse strain. Adult control mice exposed to Sugen5416 (Su)/hypoxia treatment to induce PH had significantly increased right ventricular systolic pressures (RVSP) and RV hypertrophy versus normoxic controls. In contrast, Su/hypoxia-exposed VSM ROCK1 KO mice did not exhibit significant RVSP elevation, and RV hypertrophy was blunted. Su/hypoxia-induced pulmonary small vessel muscularization was similarly elevated in both control and VSM ROCK1 KO animals. siRNA-mediated ROCK1 knock-down (KD) in human PAH pulmonary arterial SM cells (PASMC) did not affect cell growth. However, ROCK1 KD led to reduced AKT and MYPT1 signaling in serotonin-treated PAH PASMC. The findings suggest that like VSM ROCK2, VSM ROCK1 actively contributes to PH development, but in distinction acts via nonproliferative pathways to promote hypoxemia, and thus may be a distinct therapeutic target in PH.

CDC2 Is an Important Driver of Vascular Smooth Muscle Cell Proliferation via FOXM1 and PLK1 in Pulmonary Arterial Hypertension.

A key feature of pulmonary arterial hypertension (PAH) is the hyperplastic proliferation exhibited by the vascular smooth muscle cells from patients (HPASMC). The growth inducers FOXM1 and PLK1 are highly upregulated in these cells. The mechanism by which these two proteins direct aberrant growth in these cells is not clear. Herein, we identify cyclin-dependent kinase 1 (CDK1), also termed cell division cycle protein 2 (CDC2), as having a primary role in promoting progress of the cell cycle leading to proliferation in HPASMC. HPASMC obtained from PAH patients and pulmonary arteries from Sugen/hypoxia rats were investigated for their expression of CDC2. Protein levels of CDC2 were much higher in PAH than in cells from normal donors. Knocking down FOXM1 or PLK1 protein expression with siRNA or pharmacological inhibitors lowered the cellular expression of CDC2 considerably. However, knockdown of CDC2 with siRNA or inhibiting its activity with RO-3306 did not reduce the protein expression of FOXM1 or PLK1. Expression of CDC2 and FOXM1 reached its maximum at G1/S, while PLK1 reached its maximum at G2/M phase of the cell cycle. The expression of other CDKs such as CDK2, CDK4, CDK6, CDK7, and CDK9 did not change in PAH HPASMC. Moreover, inhibition via Wee1 inhibitor adavosertib or siRNAs targeting Wee1, Myt1, CDC25A, CDC25B, or CDC25C led to dramatic decreases in CDC2 protein expression. Lastly, we found CDC2 expression at the RNA and protein level to be upregulated in pulmonary arteries during disease progression Sugen/hypoxia rats. In sum, our present results illustrate that the increased expression of FOXM1 and PLK1 in PAH leads directly to increased expression of CDC2 resulting in potentiated growth hyperactivity of PASMC from patients with pulmonary hypertension. Our results further suggest that the regulation of CDC2, or associated regulatory proteins, will prove beneficial in the treatment of this disease.

Adults with cerebral palsy rank factors associated with quality of life and perceived impact of childhood surgery on adult outcomes.

BACKGROUND: Person-centered care concerns the values and perceptions of the patient in assessment of outcomes. Little is known about perceptions of adults with cerebral palsy (CP) concerning: the most important factors associated with quality of life, definitions of success, short- and long-term outcomes of childhood orthopedic care, and current needs for information surrounding childhood orthopedic care. METHODS: An online survey gathered information using structured and semi-structured questions. Descriptive statistics and content analysis summarized findings of structured questions and comments. RESULTS: A total of 71 adults with CP (44 women, median age range 30-39 years), with a variety of functional abilities, participated in the study. Priorities for a good life were clustered. There existed two groups: achievement orientation and relationship orientation. Good health and interpersonal relationships were priorities for both groups. Definitions of success included happiness, independence, meaningful relationships/activities, and mindfulness/lifelong-learning. Fifty-eight percent perceived a positive impact of childhood orthopedic care on current function. A positive perception was associated with being included in the decision making process (chi-square 25, p < .001). Suggestions to improve childhood orthopedic care included: more information about long-term outcomes, surgical timing, and alternatives to surgery, and improved service delivery models to prevent gaps in care across the lifespan. CONCLUSION: Ongoing conversations about how improvements from orthopedic surgery in childhood may not last through adolescence or adulthood are needed. The importance of impairment-based interventions should be contextualized within needs for health promotion and social engagement long-term. More research is needed on cost-benefit of childhood orthopedic surgery.Implications for RehabilitationPerceptions of being included in decisions about surgery as a child was associated with long term satisfaction.Consumer education is needed about how improvements gained in childhood may not persist through adolescence or into adulthood.Individual priorities for quality of life vary; and these priorities should be considered when weighing the cost benefit ratio of interventions.Clinicians should broaden the conversation to include how orthopedic surgery and rehabilitation afterwards could potentially impact health and well being in the future.

Participation of PLK1 and FOXM1 in the hyperplastic proliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension.

Vascular smooth muscle cells from the pulmonary arteries (HPASMC) of subjects with pulmonary arterial hypertension (PAH) exhibit hyperplastic growth. The PAH HPASMC display an increased sensitivity to fetal bovine serum (FBS) and undergo growth at a very low, 0.2%, FBS concentration. On the other hand, normal HPASMC (obtained from non-PAH donors) do not proliferate at low FBS (0.2%). A previous genomic study suggested that the nuclear factor, FOXM1 and the polo like kinase 1 (PLK1) are involved in promoting this hyperplastic growth of the PAH HPASMC. Here we find that limiting the action of FOXM1 or PLK1 not only restricts the hyperplastic proliferation of the PAH HPASMC but also modulates the FBS stimulated growth of normal HPASMC. The PAH HPASMC exhibit significantly elevated PLK1 and FOXM1 expression and decreased p27 (quiescence protein) levels compared to normal HPASMC. Regulation of the expression of FOXM1 and PLK1 is accompanied by the regulation of downstream expression of cell cycle components, Aurora B, cyclin B1 and cyclin D1. Expression of these cell cycle components is reversed by the knockdown of FOXM1 or PLK1 expression/activity. Furthermore, the knockdown of PLK1 expression lowers the protein level of FOXM1. On the other hand, inhibiting the action of FOXO1, a growth inhibitor, further increases the expression of FOXM1 in PAH HPASMC. Although PLK1 and FOXM1 clearly participate in PAH HPASMC hyperplasia, at this time it is not clear whether their increased activity is the primary driver of the hyperplastic behavior of the PAH HPASMC or merely a component of the pathway(s) leading to this response.

Unraveling endothelin-1 induced hypercontractility of human pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension.

Contraction of human pulmonary artery smooth muscle cells (HPASMC) isolated from pulmonary arterial hypertensive (PAH) and normal (non-PAH) subject lungs was determined and measured with real-time electrical impedance. Treatment of HPASMC with vasoactive peptides, endothelin-1 (ET-1) and bradykinin (BK) but not angiotensin II, induced a temporal decrease in the electrical impedance profile mirroring constrictive morphological change of the cells which typically was more robust in PAH as opposed to non-PAH cells. Inhibition with LIMKi3 and a cofilin targeted motif mimicking cell permeable peptide (MMCPP) had no effect on ET-1 induced HPASMC contraction indicating a negligible role for these actin regulatory proteins. On the other hand, a MMCPP blocking the activity of caldesmon reduced ET-1 promoted contraction pointing to a regulatory role of this protein and its activation pathway in HPASMC contraction. Inhibition of this MEK/ERK/p90RSK pathway, which is an upstream regulator of caldesmon phosphorylation, reduced ET-1 induced cell contraction. While the regulation of ET-1 induced cell contraction was found to be similar in PAH and non-PAH cells, a key difference was the response to pharmacological inhibitors and to siRNA knockdown of Rho kinases (ROCK1/ROCK2). The PAH cells required much higher concentrations of inhibitors to abrogate ET-1 induced contractions and their contraction was not affected by siRNA against either ROCK1 or ROCK2. Lastly, blocking of L-type and T-type Ca2+ channels had no effect on ET-1 or BK induced contraction. However, inhibiting the activity of the sarcoplasmic reticulum Ca2+ ATPase blunted ET-1 and BK induced HPASMC contraction in both PAH and non-PAH derived HPASMC. In summary, our findings here together with previous communications illustrate similarities and differences in the regulation PAH and non-PAH smooth muscle cell contraction relating to calcium translocation, RhoA/ROCK signaling and the activity of caldesmon. These findings may provide useful tools in achieving the regulation of the vascular hypercontractility taking place in PAH.

Hyperplastic Growth of Pulmonary Artery Smooth Muscle Cells from Subjects with Pulmonary Arterial Hypertension Is Activated through JNK and p38 MAPK.

Smooth muscle in the pulmonary artery of PAH subjects, both idiopathic and hereditary, is characterized by hyperplasia. Smooth muscle cells (HPASMC) isolated from subjects with or without PAH retain their in vivo phenotype as illustrated by their expression of alpha-smooth muscle actin and expression of H-caldesmon. Both non PAH and PAH HPASMC display a lengthy, approximately 94h, cell cycle. The HPASMC from both idiopathic and hereditary PAH display an abnormal proliferation characterized by continued growth under non-proliferative, non-growth stimulated conditions. This effector independent proliferation is JNK and p38 MAP kinase dependent. Blocking the activation of either abrogates the HPASMC growth. HPASMC from non PAH donors under quiescent conditions display negligible proliferation but divide upon exposure to growth factors such as PDGF-BB or FGF2 but not EGF. This growth does not involve the MAP kinases. Instead it routes via the tyrosine kinase receptor through mTOR and then 6SK. In the PAH cells PDGF-BB and FGF2 augment the dysregulated cell proliferation, also through mTOR/6SK. Additionally, blocking the activation of mTOR also modulates the MAP kinase promoted dysregulated growth. These results highlight key alterations in the growth of HPASMC from subjects with PAH which contribute to the etiology of the disease and can clearly be targeted at various regulatory points for future therapies.

Targeting receptor tyrosine kinases and their downstream signaling with cell-penetrating peptides in human pulmonary artery smooth muscle and endothelial cells.

Cell-penetrating peptide (CPP) intracellular delivery of receptor signaling motifs provides an opportunity to regulate specific receptor tyrosine kinase signal transductions. We targeted tyrosine residues Y740 and Y751 of the PDGF receptor ß (PDGFRß) and Y1175 of the VEGF receptor 2 (VEGFR2). The Y740 and Y751 motifs activated ERK and Akt, while the Y1175 motif activated ERK. Targeting either Y740 or Y751 of the PDGFRß in human pulmonary artery smooth muscle cells (HPASMC) effectively inhibited PDGF activation of ERK or Akt. Interfering with the Y751 region of the PDGFRß proved more effective than targeting the Y740 region. The phosphorylation of Y751 of the CPP and the length and exact sequence of the mimicking peptide proved crucial. On the other hand, in human pulmonary artery endothelial cell phosphorylation of the VEGFR2 Y1175 CPP was not a determinant in blockage of ERK activation. Likewise, the length of the peptide mimic was not crucial with a very small sequence containing the Y1175 remaining effective. Physiologic proof of concept for the effectiveness of the CPP was confirmed by blockage of HPASMC migration in response to PDGF following culture injury. Thus targeted blockage of tyrosine kinase receptor signaling can be very effective.

Modulating the dysregulated migration of pulmonary arterial hypertensive smooth muscle cells with motif mimicking cell permeable peptides.

Migration of vascular smooth muscle cells is a key element in remodeling during pulmonary arterial hypertension (PAH). We are observing key alterations in the migratory characteristics of human pulmonary artery smooth muscle cells (HPASMC) isolated from transplanted lungs of subjects with PAH. Using wound migration and barrier removal assays, we demonstrate that the PAH cells migrate under quiescent growth conditions and in the absence of pro-migratory factors such as platelet derived growth factor (PDGF). Under the same conditions, in the absence of PDGF, non-PAH HPASMC show negligible migration. The dysregulated migration initiates, in part, through phosphorylation events signaled through the unstimulated PDGF receptor via focal adhesion kinase (FAK) whose total basal expression and phosphorylation at tyrosine 391 is markedly increased in the PAH cells and is inhibited by a motif mimicking cell-permeable peptide (MMCPP) targeting the Tyr751 region of the PDGF receptor and by imatinib. However, exposure of the PAH cells to PDGF further promotes migration. Inhibition of p21 activated kinases (PAK), LIM kinases (LIMK), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) reduces both the dysregulated and the PDGF-stimulated migration. Immunofluorescence microscopy confirms these observations showing activated JNK and p38 MAPK at the edge of the wound but not in the rest of the culture in the PAH cells. The upstream inhibitors FAK (PF-573228) and imatinib block this activation of JNK and p38 at the edge of the site of injury and correspondingly inhibit migration. MMCPP which inhibit the activation of downstream effectors of migration, cofilin and caldesmon, also limit the dysregulated migration. These results highlight key pathways which point to potential targets for future therapies of pulmonary hypertension with MMCPP.

A cell permeable peptide targeting the intracellular loop 2 of endothelin B receptor reduces pulmonary hypertension in a hypoxic rat model.

Cell permeable peptides (CPP) aid cellular uptake of targeted cargo across the hydrophobic plasma membrane. CPP-mediated cargo delivery of receptor signaling motifs provides an opportunity to regulate specific receptor initiated signaling cascades. Both endothelin-1 receptors, ETA and ETB, have been targets of antagonist therapies for individuals with pulmonary arterial hypertension (PAH). These therapies have had success but have been accompanied by adverse reactions. Also, unlike the CPP which target specific signaling cascades, the antagonists target the entire function of the receptor. Using the CPP strategy of biased antagonism of the ETB receptor's intracellular loop 2 (ICB2), we demonstrate blunting of hypoxic pulmonary hypertension (HPH) in the rat, including indices of pulmonary arterial pressure, right ventricular hypertrophy and pulmonary vascular remodeling. Further, ex vivo analysis of the pulmonary artery treated with the IC2B peptide upon injection manifests marked reductions in Akt and ERK activation. Both kinases have been intimately related to cell proliferation and vascular contraction, the hallmarks of PAH. These observations in sum illustrate an involvement of the ETB receptor in HPH and furthermore provide a basis for a novel, CPP-based, strategy in the treatment of PAH, ultimately able to target not only ET-1, but also other factors involved in the development of PAH.

C-terminus of ETA/ETB receptors regulate endothelin-1 signal transmission.

The dimerization of the G protein-coupled receptors for endothelin-1 (ET-1), endothelin A receptor (ETA) and endolethin B receptor (ETB), is well established. However, the signaling consequences of the homodimerization and heterodimerization of ETA and ETB is not well understood. Here, we demonstrate that peptides derived from the C-termini of these receptors regulate the signaling capacity of ET-1. The C-termini of the ETA and ETB receptors are believed to consist of three α-helices, which may serve as points of interaction between the receptors. The third α-helix in the C-terminus is of particular interest because of its amphipathic nature. In a cell line expressing only the ETA receptor, expression of residues Y430-S442, representing the third helix of the ETB C-terminus, leads to a dramatic increase in the signaling induced by ET-1. In contrast, in a cell line containing only ETB , Y430-S442 has an antagonistic effect, slightly reducing the ET-1 induced signal. Computational docking results suggest that the α-helical ETB -derived peptide binds to the second and third intracellular loops of the ETA receptor consistent with the alteration of its signaling capacity. Our results described here provide important insight into ETA /ETB receptor interactions and possibly a new approach to regulate specific G protein-coupled receptor signal transmission.

Forensic analysis of mtDNA haplotypes from two rural communities in Haiti reflects their population history.

Very little genetic data exist on Haitians, an estimated 1.2 million of whom, not including illegal immigrants, reside in the United States. The absence of genetic data on a population of this size reduces the discriminatory power of criminal and missing-person DNA databases in the United States and Caribbean. We present a forensic population study that provides the first genetic data set for Haiti. This study uses hypervariable segment one (HVS-1) mitochondrial DNA (mtDNA) nucleotide sequences from 291 subjects primarily from rural areas of northern and southern Haiti, where admixture would be minimal. Our results showed that the African maternal genetic component of Haitians had slightly higher West-Central African admixture than African-Americans and Dominicans, but considerably less than Afro-Brazilians. These results lay the foundation for further forensic genetics studies in the Haitian population and serve as a model for forensic mtDNA identification of individuals in other isolated or rural communities.

Enhancing and limiting endothelin-1 signaling with a cell-penetrating peptide mimicking the third intracellular loop of the ETB receptor.

TAT (a 13-mer derived from the HIV-1 Tat protein)-linked cell-permeable peptides deliver plasma membrane impermeable cargos into the cell. We investigated the effect of a TAT-linked intracellular third loop of the endothelin-1 type B receptor on endothelin-1 activation of ERK. The effect of this peptide on ERK activation was determined in ETB receptor cDNA-transfected Chinese hamster ovary cells and in ETA- and ETB-expressing human pulmonary artery smooth muscle cells obtained from a normal and a bone morphogenetic protein-2 receptor, exon 1-8 deletion subject, with pulmonary hypertension. In the Chinese hamster ovary cells the peptide, at optimum 10 µm concentration, suppressed endothelin-1 activation. In the normal human pulmonary artery smooth muscle cells, the peptide marginally enhanced endothelin-1 activation of ERK. However, it markedly enhanced the endothelin-1 activation of ERK in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells. While the effective concentration for endothelin-1 activation of ERK remained unchanged in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells, the number of ETB receptors declined by 2/3. These data point to the intracellular third loop peptide as having variable receptor interactive effects with both signal repressive and enhancing capabilities. Peptides that can alter endothelin-1 signal capabilities are potentially important in the study and treatment of pulmonary hypertension.

Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint.

Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [(3)H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension.